【摘要】：血吸蟲病是免疫性疾病,血吸蟲性別特異分化、生殖產(chǎn)卵等階段對其生存?zhèn)鞑ビ兄匾绊憽１菊n題組前期篩選出幾十個可能與日本血吸蟲免疫、性別及生殖相關(guān)的蛋白,作為日本血吸蟲診斷抗原與疫苗的候選靶標(biāo)。本研究針對其中的SjTollip(AY814053, AAW25785.1)和SjSSX(BU793503, CAX76185.1)2個蛋白進(jìn)行研究。主要研究內(nèi)容包括：(1)根據(jù)序列信息進(jìn)行以下生物信息學(xué)分析：預(yù)測信號肽、預(yù)測跨膜區(qū)域、預(yù)測功能位點和結(jié)構(gòu)域、同源序列搜索、多序列比對以及構(gòu)建進(jìn)化樹等；(2)基因的克隆、原核表達(dá)以及重組蛋白的純化；(3)重組蛋白免疫原性檢測,制備重組蛋白兔多克隆抗體并評價,考察基因的蟲期轉(zhuǎn)錄特異性和蛋白的蟲期表達(dá)特異性；(4)蛋白質(zhì)的診斷價值評價和動物保護(hù)性實驗。 Tollip是TLRs信號通路中的負(fù)性調(diào)節(jié)因子,它能夠抑制過度和長時間的免疫反應(yīng)。SjTollip為相對保守蛋白,在多個物種中有同源序列。構(gòu)建了pET-28a/SjTollip重組質(zhì)粒,誘導(dǎo)表達(dá)獲得不可溶蛋白,通過尿素純化獲得較高濃度和純度的重組蛋白；Western blot檢測顯示SjTollip具有一定的免疫原性；SjTollip免疫兔多克隆抗體的滴度達(dá)到1：256,000；SjTollip在毛蚴、胞蚴、蟲卵、童蟲、雌蟲中轉(zhuǎn)錄水平高,尾蚴和雄蟲中轉(zhuǎn)錄水平低；SjTollip主要在童蟲和雌蟲中表達(dá),與基因轉(zhuǎn)錄一致；rSjTollip作為包被抗原通過ELISA方法檢測不同人群血清,敏感性和特異性分別為70%和80%；用rSjTollip免疫小鼠誘導(dǎo)產(chǎn)生58.05%的減蟲率,感染后40d-42d糞便減卵率為59.84%,43d～45d糞便減卵率為78.68%。說明其能夠誘導(dǎo)產(chǎn)生較強(qiáng)的免疫保護(hù)力,是潛在的疫苗候選靶標(biāo)。 SSX蛋白是CT抗原家族成員,CT抗原的異位表達(dá)可以導(dǎo)致自體統(tǒng)一細(xì)胞或體液產(chǎn)生免疫應(yīng)答,可用于構(gòu)建和發(fā)展免疫治療方案,作為免疫治療的靶點。SjSSX與日本血吸蟲重要宿主中的同源序列的相似性較低,構(gòu)建了pET-28a/SjSSX重組質(zhì)粒,誘導(dǎo)表達(dá)獲得不可溶蛋白,通過尿素純化獲得較高濃度和純度的重組蛋白；Western blot檢測顯示SjSSX具有良好的免疫原性；SjSSX免疫兔多克隆抗體的滴度達(dá)到1：256,000;SjSSX在童蟲、雌蟲中轉(zhuǎn)錄水平高,胞蚴、蟲卵次之,毛蚴、尾蚴和雄蟲中轉(zhuǎn)錄水平低；SjSSX在蟲卵、尾蚴、童蟲、雄蟲、雌蟲均有表達(dá)；SjSSX作為包被抗原通過ELISA方法檢測不同人群血清,敏感度為80%,特異度為90%；用SjSSX免疫小鼠可誘導(dǎo)產(chǎn)生28.16%的減蟲率,感染后43d-45d糞便減卵率為4.25%。
[Abstract]:Schistosomiasis is an immune disease. Sex specific differentiation of schistosomiasis and reproductive oviposition play an important role in the survival and transmission of schistosomiasis. Dozens of proteins that may be related to Schistosoma japonicum immunity, sex and reproduction were screened by our research group, which were used as candidate targets for diagnostic antigen and vaccine of Schistosoma japonicum. In this study, two proteins, SjTollip (AY814053, AAW25785.1) and SjSSX (BU793503, CAX76185.1), were studied. The main contents are as follows: (1) the following bioinformatics analysis is carried out according to the sequence information: predicting signal peptide, predicting transmembrane region, predicting functional site and domain, homologous sequence searching, multi-sequence alignment and constructing evolutionary tree; (2) cloning of gene, prokaryotic expression and purification of recombinant protein; (3) immunogenicity of recombinant protein, preparation and evaluation of rabbit polyclonal antibody against recombinant protein; (4) the diagnostic value of protein and animal protective experiment. Tollip is a negative regulatory factor in TLRs signaling pathway, which can inhibit excessive and long-term immune response. SjTollip is a relatively conserved protein with homologous sequences in many species. The recombinant plasmid of pET-28a/SjTollip was constructed, and the insoluble protein was induced to express. The high concentration and purity of recombinant protein; Western blot were obtained by urea purification. The results showed that SjTollip had certain immunogenicity. The titer of polyclonal antibody of rabbit immunized with SjTollip reached 1: 256000 Sj Tollip with high transcription level in cercariae, cystis, eggs, juvenile worms, female worms, and low transcription levels in cercariae and males, and SjTollip was mainly expressed in juvenile and female worms, which was consistent with gene transcription. The sensitivity and specificity of rSjTollip were 70% and 80%, respectively. The worm reduction rate of rSjTollip immunized mice was 58.05%, and the fecal egg reduction rate of 40d-42d was 59.84 days after infection. The fecal egg reduction rate was 78.68 days after infection. It can induce strong immune protection and is a potential vaccine candidate target. SSX protein is a member of CT antigen family. The heterotopic expression of CT antigen can lead to the immune response of autologous uniform cells or humoral fluids, and can be used to construct and develop immunotherapy protocols. As a target of immunotherapy, the homologous sequence of SjSSX and Schistosoma japonicum host was low. The recombinant plasmid of pET-28a/SjSSX was constructed and induced to express insoluble protein. High concentration and purity of recombinant protein were obtained by urea purification. Western blot analysis showed that SjSSX had good immunogenicity, the titer of SjSSX immunized rabbit polyclonal antibody reached 1: 256000, and the transcription level of SjSSX was higher in chicks and females, and the transcription level was lower in cercariae, cercariae and male worms than in cystis, eggs, cercariae and males. SjSSX was expressed in egg, cercariae, child worm, male worm, female worm, SjSSX was used as coating antigen to detect serum of different population by ELISA method, sensitivity was 80, specificity was 90. Mice immunized with SjSSX produced 28.16% worm reduction rate and 4.25% egg reduction rate in 43d-45d feces after infection.
【學(xué)位授予單位】：華東理工大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2011
【分類號】：R392.1

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